Project Summary/Abstract Alpha-1 antitrypsin (AAT) is the major circulating serum antiprotease, with normal plasma levels ranging from 11 to 30 microMolar. Approximately 4% of the North American and Northern European populations possess at least one copy of a mutant allele, known as Pi*Z, which results from a single amino acid substitution of Lys for Glu at position 342. In the homozygous state (Pi*ZZ), this mutation leads to severe deficiency of AAT and can result in liver disease which is due to a toxic gain-of-function of the Z-AAT mutant protein. Accumulation of Z- AAT polymers within hepatocytes consistently results in a state of serum AAT deficiency due to inefficient secretion. Two hundred thousand is a conservative estimate of the number of ZZ patients in the western world; however, there is currently no cure for the lung or liver disease. Protein replacement is given by intravenous infusion, with the approved dosing regimen being weekly infusions, and the only alternative dosing with proven equivalence being every-other-week infusions. Thus, gene augmentation with normal (PiM) AAT delivered via recombinant adeno- associated viral (rAAV) vectors has been developed as a potential alternative for ?sustained release? of the therapeutic protein. We believe that in order to safely augment AAT from Z-AAT patient livers it may be necessary to silence the endogenous Z-AAT to prevent overwhelming the already stressed hepatocyte. Preliminary data in PiZ transgenic mice suggest that expressing rAAV-derived AAT without first silencing the endogenous Z-AAT can be hepatotoxic. Thus, this project aims to clinically translate a therapeutic strategy that combines traditional gene therapy with RNAi therapeutics in a single rAAV vector approach. We have termed these as dual-function rAAV vectors, and we have engineered them as a ?liver- sparing? approach to target the burdened livers of PiZ AATD patients. Proof-of-concept studies in PiZ mice, confirmed that the dual function vector reduced misfolded AAT and concomitantly augmented serum levels of the normal protein. We hypothesize that endogenous Z-AAT silencing and M-AAT augmentation is feasible and a safe therapeutic approach for treating alpha-1 antitrypsin deficiency. We will test this hypothesis with the following three aims. In aim 1 we will optimize the dual function expression cassette for maximum knockdown and augmentation efficiency. These vectors will then be modified with non- human primate sequences (NHP) for aim 2, where we will assess efficacy of the dual function vector in non- human primates. In aim 3 we will perform a formal GLP pharm/tox and biodistribution study with the clinical version of the vector to prepare for an investigational new drug application with the FDA. Finally aim 4 will investigate the safety of peripheral intravenous delivery of these vectors in patients with alpha-1 antitrypsin deficiency.